Fig. 1

XIST-mediated silencing depends on Repeats A and F as well as 3’ non-repeat domain. A The change in allelic contribution over time of four 8p genes repressed by the induction of the XIST cDNA construct in the HT1080 cell line are shown. Three biological replicates were tested for each time point and significance was calculated using a student t-test relative to 5ddox. B The change in allelic contribution (for the same genes as in A) caused by 5 days of induction of XIST deletion constructs are shown normalized to the full-length XIST construct. A value of 1 indicates that the strength of silencing was comparable to Full XIST, a value of 0 indicates no silencing occurred. Deletion constructs where all 4 alleles statistically differed from Full XIST by t-test with multiple testing correction are indicated. C FISH images of XIST (green) and Cot-1 (red) and DAPI (blue) used to exemplify the formation of a transcriptionally inert “Cot-1 hole” in Full XIST HT1080 cells treated with dox for 5 days. D The depletion of Cot-1 RNA at the site of XIST RNA was calculated as the standard score (z-score) for each of the 60 cells examined at 2, 5 and 10 days of induction. Cell populations were blinded through the process and the distribution of conditions were statistically compared using a Mann–Whitney test with multiple testing correction. There was no significant change across the timepoints. E Each deletion construct induced for 5 days was tested for its relative depletion (z-score) of Cot-1 RNA hybridization at the site of XIST. Samples were blinded and statistically compared to Full XIST using a Mann–Whitney test with multiple testing correction. A–E The thresholds for statistical significance of all p values are: * < 0.05, ** < 0.01 and *** < 0.001, with these thresholds adjusted based on the number of tests